The overall goal of this translational program is to develop a definitive molecular therapy for lung disease due to alpha-1 antitrypsin (AAT) deficiency, a relatively common single gene disease due to mutations in the AAT gene. The program builds on the experience of investigators in developing several different versions of recombinant adeno-associated virus (rAAV)-based gene therapies for AAT deficiency since the late 1990's. Previous NHLBI-funded work by the program director's laboratory included proof-of-concept (POC) studies, formal IND-enabling safety studies, and a phase 1 trial of intramuscular injection of a 1st generation rAAV2- AAT (on a physician IND). Subsequently, the same gene cassette was packaged in rAAV1-AAT for IM injection (2nd generation rAAV1-AAT) was likewise studied through POC, IND-enabling and phase 1 and phase 2a studies in a public-private partnership between NHLBI funding and funding by a small biotechnology company (AGTC). These studies showed serum AAT expression persisting for several years at level only 30-fold below the therapeutic threshold and demonstrated an important role for the induction of regulatory T cells (Tregs) in these patients. In the coming proposal, a group of investigators within the UMMS Horae Gene Therapy Center (GTC) and the RNA Therapeutics Institute (RTI) have come together to push translational studies of rAAV-AAT of the 2nd generation vector along with a number of newer approaches. These newer approaches include innovative RNA-based therapeutics, such as synthetic miRNAs (3rd generation), CRISPR/Cas9 approaches (4th generation), and novel AAV capsids (Nth generation). Additional innovation and accelerated translation is provided by superb research cores, including a transgenic animal core that has used the CRISPR/Cas9 system to create an AAT null mouse, respiratory physiology core to define the emphysema phenotype in these mice, and a vector core that will both innovate and provide high quality vector for all projects. With these successive generations of vectors, this program seeks to address the unmet need for gene therapy for lung disease due to alpha-1 antitrypsin (AAT) deficiency with an important focus on liver-sparing systemic gene therapy, designed to treat AAT lung disease without exacerbating AAT liver disease. Narrative: This proposal seeks to use the most cutting edge scientific techniques in gene therapy to develop a way to treat an inherited lung disease, called AAT deficiency. This disease leads to a form chronic obstructive pulmonary disease (COPD) and emphysema, which overall affect over 11 million patients in the US. AAT deficiency is less common than non-inherited forms of COPD, with estimates ranging from approximately 10,000 to 100,000 cases in the US, with many cases going undiagnosed. PROJECT 1: Clinical Trial and Immunologic aspects of muscle-directed rAAV1-AAT gene Therapy (Flotte, Terence R., M.D.)